Internally reflective chamber for flourescent radiation collection and concentration, and method for using the same

ABSTRACT

A system for collecting and analyzing maximized amounts of fluorescent radiation using frequency scattering ports or waveguides that absorb a desired size of wavelengths.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to co-pending U.S. provisional patentapplication Ser. No. 61/736,749, filed Dec. 13, 2012; and co-pendingU.S. provisional patent application Ser. No. 61/755,165, filed Jan. 22,2013, each of which are incorporated herein in their entirety byreference.

TECHNICAL FIELD

The present novel technology relates to the field of laser physics, and,more particularly, to a laser-based cytometer flow measurement system.

BACKGROUND

In the field of cytometry, collection and analysis of fluorescentradiation is important. Cells or particles of interest are bound withvarious fluorescent tags and generally sent via fluidic transportthrough an interrogation point, where the particles are then illuminatedsuch as by a laser or other light source. Given an appropriate tag thatwill interact with the incident wavelength of light, the particles willthen radiate a fluorescent signal that indicates a particular trait heldby the particles in interest. This signal is then processed through anoptical train, typically consisting of a combination of lenses, fibersand/or dichroic mirrors to relay the fluorescent signal to a finaldetector to be captured. Overall, flow cytometers collect a relativelysmall amount of the omitted fluorescent signal. This weak signalnecessitates the use of photomultiplier tubes (PMTs) for the directmeasurement of the fluorescent signal, necessitating yet furtheramplification of the PMT's output for subsequent samplecharacterization. The desire to increase the amount of fluorescentradiation collected is very great, and has generated considerable workand various approaches to reaching this elusive goal. Simply stated,increasing the amount of fluorescent radiation collected for analysiswill allow lower threshold levels of radiation to be measured, therebyboosting the number of particles in interest observed and increasing theprobability of detecting sporadic or rare events held within the sampleset. Conventional commercial flow cytometers typically utilize highnumerical aperture optics to collect the fluorescent radiation foranalysis. This technique limits the amount of radiation that can becollected by constraining the volume of the fluorescence emitted forobservation to that of the numerical aperture optics used. If there wasa way to collect and analyze a greater amount of the fluorescentradiation signal, it would be possible to better track the presence ofcertain cells in health or cancer research, or increase accuracy whenconducting research into biomarkers, and gain better feedback forprotein engineering, and the like. Thus, a need persists for a moreeffective technique for capturing and utilizing a greater portion of thegenerated fluorescent signal during cytometric analysis. The presentnovel technology addresses this need.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic side sectional view of the internally reflectivechamber for fluorescent radiation collection and concentration,according to the first embodiment of the present novel technology.

FIG. 2 is a schematic front sectional view of the internally reflectivechamber for fluorescent radiation collection and concentration, of FIG.1.

FIG. 3 is a schematic side sectional view of a solid externallyreflective flow cell for the collection and concentration of fluorescentradiation, according to the second embodiment of the present noveltechnology.

FIG. 4 is a diagram of the solid externally reflective flow cell for thecollection and concentration of fluorescent radiation.

FIG. 5 is a schematic view of a spherical florescent radiation detectorcell, of the present novel technology.

FIG. 6 is a schematic diagram of a multispectral wavelength selectivedetector, for use with the present novel technology.

FIG. 7 is a schematic diagram of the pixelated architecture of themultispectral wavelength selective detector of FIG. 6.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of thenovel technology and presenting its currently understood best mode ofoperation, reference will now be made to the embodiments illustrated inthe drawings and specific language will be used to describe the same. Itwill nevertheless be understood that no limitation of the scope of thenovel technology is thereby intended, with such alterations and furthermodifications in the illustrated device and such further applications ofthe principles of the novel technology as illustrated therein beingcontemplated as would normally occur to one skilled in the art to whichthe novel technology relates.

In traditional cytometer flow cell architecture, a very small percentageof the fluorescent radiation generated by the sample being investigatedis collected usefully. The novel technology presented herein relates toa method and apparatus for the collection of most or virtually theentire radiation signal generated by fluorescing a sample materialand/or maximizes the collected signal to increase detectioncapabilities. The novel technology operates to encase the point offluorescence in a hollow reflective chamber wherein the emittedradiation is redirected and/or concentrated for measurement andanalysis. The basic configuration from an optical componentmanufacturing or light manipulation standpoint is typically sphericallybased, more typically an integration sphere or partial sphere to encasethe point of fluorescence. To concentrate and direct the fluorescentradiation emitted during cytometry, the integration sphere is typicallytruncated to become a highly reflective hemisphere and then conjoinedwith a high collection, non-imaging reflective optical component with asingular circular output. To increase collection and light concentrationefficiency, the profile of this non-imaging reflective component istypically determined to be a hyperboloid of revolution, as this shapelends itself to useful radiation concentration. As an added benefit, thelight emitted from the hyperboloid of revolution is very well-behavedand lends itself nicely to manipulation and transmission. This designand functionality applies to either the hollow core or opticallytransparent solid core versions of the flow cell chamber.

While fluorescence measurement is one useful aspect of flow cytometryanalysis, forward scatter (FS) and side scatter (SS) measurements may beequally useful. The design disclosed herein also addresses thesemeasurements as well. Dependent upon the particular version of thechamber selected, ports may be either machined directly into the chamber(hollow core) or machined into the lower index coating material (solidcore) such that the traditional methods of FS and SS measurements stillapply.

One embodiment of the novel technology as illustrated in FIG. 1 and FIG.2 is a flow cytometer 1 having an internally reflective, generallyhollow chamber 5 in which a sample may be laser fluoresced with thefluorescent radiation collected and concentrated. The chamber 5typically either inherently reflective or has the shape of a hemisphereand functions as a high collection, non-imaging reflector. The chamber 5is typically internally coated 10 with broadband reflective material,the selection of which is typically substrate dependent and of a lowerindex material. A capillary transport 15 is positioned at one or morepredetermined locations within the chamber 5 to supply particles 40 froma flow cell 20, which is typically positioned outside the chamber 5,through a focus position 25 of an operationally connected integrationlaser 30, likewise typically positioned outside the cell. The focuspotion 25 is typically centrally positioned inside the chamber 5.Frequency scattering ports 35 are typically positioned around thechamber 5 to facilitate laser focus as well as forward and side scatterdetection. The ports 35 for laser 30 introduction and the forwardscatter measurement typically lie in a line that intersects the focusposition 25 of the hemisphere. The side scatter port 35 and focalposition 25 of the hemisphere 5 typically lie in a line orthogonal tothe line formed by the laser(s) (or light) port, focal point of thehemisphere 5, and the forward scatter port. Particles emitted from theparticle interrogation position 25 are collected by reflection andemitted out of the non-imaging reflector output portion 45. The newimaging reflector output portion 45 is operationally connected inphotonic communication with the hemisphere chamber 5 to guide andconcentrate fluoresced signals from the particles to an output port 47.The output portion 45 typically has the shape of a hyperboloid ofrevolution, although it may have any convenient shape. The collectedparticles 40 is then evaluated according to methods commonly used in theart, such as spectrometry or other like means of evaluation ormeasurement. Fiber optic conduits 50 may also be used to transport theinput or output of particles 40 of the chamber 5. Additional observationports 35 may be added to the chamber 5 to allow further imaging of theinterrogation point 25.

A second embodiment of the instant novel technology, as illustrated inFIG. 3 and FIG. 4, is a flow cytometry system 55 having an externallyreflective chamber 60. The solid, optically transparent externallyreflective chamber 60 is typically a unitary combination of anintegration hemisphere 67 made of a high collection, non-imagingreflector-material selected for broad wavelength transmission. Thechamber 60 is typically externally coated 65 for broadband internalreflection. The chamber 60 includes a generally culcindrical elongatedconduit 61 formed there through. A capillary transport 70 extendsthrough the conduit 61 within the chamber 60 to supply particles 90 froma flow cell 75, which is positioned outside the chamber 60 and influidic communication with the capillary transport system 70, through afocus position 80 of an operationally connected integration laser 85,likewise typically positioned outside the cell. The focus position 80 istypically strategically positioned inside the conduit 61. Frequencyscattering ports 95 through the coating 65 are located at thepreselected positions on the hemisphere 67 to allow laser 85 focus aswell as forward and side scatter detection. The ports 95 for laser 85introduction and the forward scatter measurement lie in a line thatcontains the focal point 80 of the hemisphere 87. The side scatter portand focal point 80 of the hemisphere 87 typically lie in a intersectingthe laser 85 port, focal point 80 of the hemisphere 87, and the forwardscatter port 95. Particles 90 emitted from the particle interrogationposition 80 are collected by reflection and emitted out of thenon-imaging reflector output positions 100. The collected particles 90may then be evaluated though methods commonly used in the art such as aspectrometer or other means of evaluation or measurement. As with theprevious embodiment, fiber optic conduits 50 may also be used totransport the particle 90 input or output of the chamber 60. An exampleof a possible coating 10,65 material for the first and second embodimentwould be aluminum, which may increase reflectivity to at least 60% moretypically at least 70%, still more typically at least 80%, and even upto at least 90%, allowing close to the theoretical maximum amount offluorescent energy to be collected for analysis.

A third embodiment, illustrated in FIG. 5, is a flow cytometry system101 having a generally spherical reflective chamber 105. The sphericalreflective chamber 105 operates much like the above described first andsecond embodiments, but the spherical shape allows for possiblealternative observation strategies for collection of the forward andside scatter of particles 140 through the input and output ports.Similar to the first and second embodiments, capillary tubing 120provides the input and output of the flow cell 155 via a capillary 120channel through the center of the cell. The capillary transport ispositioned in the center of the spherical chamber 105, to supplyparticles 140 from a flow cell 155, which is positioned outside thechamber 105, through a focus position 125 of the integration laser 130.Frequency scattering ports 135 are typically located at the appropriatepositions on the sphere to allow laser 130 focus as well as forward andside scatter detection ports 135. The ports 150 for light beamintroduction and the forward scatter measurement typically lie in a linethat contains the focal point 125 of the sphere 137. The side scatterport 135 and focal position 125 of the hemisphere lie in a lineintersecting the laser port 150, focal point of the hemisphere 125, andthe forward scatter port 135. Optional ports can be added for directimaging of the interrogated particles 140. Particles 140 emitted fromthe particle interrogation position 125 are collected by reflection andemitted out of the non-imaging reflector output positions 145. Thecollected particles 140 can then be evaluated though methods commonlyused in the art such as a spectrometer or other means of evaluation ormeasurement. The surface of the sphere is typically covered with apixelated multispectral wavelength detector 160 to capture the particlesreleased from the chamber 105. The pixelated multispectral wavelengthdetector 160 typically encapsulates the entire spherical chamber 105 inits entirety, but for the side and forward scattering observation ports135 left clear for observation analysis. Alternatively, the chamber 105may be coated with a broadband anti-reflective coating 162, to restrainthe radiation 140 in the chamber 105, if no measuring device isencapsulating the chamber 105 for particle 140 analysis. Fiber optics 50may also be used to transport the input or output of the chamber 105.Additional observation ports 135 may be added to the chamber 105 toallow imaging of the interrogation point 125.

In any of the above embodiments, an ultrasonic transducer 300 can beused to generate ultrasonic waves 305 to guide particle 40, 90, 140 flowsuch that corresponding ultrasonic wave pressure acts as a gate allowingparticles to pass through the focal point 25, 80, 125 individually.

The particles 40, 90, 140 may be injected into the embodiment 1, 55, 101at an input point 310 between the ultrasonic transducer 300 and thefocal point 25, 80, 125. Once the particles 40, 90, 140 are aligned pastthe focal point 25, 80, 125, the separated particles 40, 90, 140 and maybe evaluated from the output ports 315 though methods commonly used inthe art such as a spectrometer or other means of evaluation ormeasurement.

A fourth embodiment useful for assisting in spectral analysis offluorescent radiation as generated and connected in the above cytometrysystems 1, 55, 101, illustrated in FIG. 6 and FIG. 7, is a multispectralwavelength selective detector 250 made of multiple layers of waveguides290. The layers of waveguides 290 are dimensionally sized to eitherallow the transmission of incident wavelengths 255, to be collected foranalysis later, or absorption of a selected wavelength 255 formeasurement. Longer wavelengths 255 are measured toward the input 265 ofthe detector, with shorter wavelengths measured by layers father intothe lights path's penetration into the detector 250. The layers 290 arecomposed of alternating insulator 27 layer, N layer 275 then P layer 280repeatedly stacked in consecutive order. Coatings may be applied to thewaveguide 290 layers to increase light transmission into the detector250. Overall waveguide 290/detector 250 construction is of a pixelatedarchitecture 285. The detector 250 can be used to encapsulate reflectivechambers, such as those described by the third embodiment, to analyzethe particles released from the chamber. This detector 250 architecturemay be applied to other measurement or observation methods forwavelength 225 analyses.

It will be appreciated that several of the above-disclosed and otherfeatures and functions, or alternatives thereof, may be desirablycombined into many other different systems or applications. Also, thatvarious presently unforeseen or unanticipated alternatives,modifications, variations or improvements therein may be subsequentlymade by those skilled in the art which are also intended to beencompassed by the following claims.

While the claimed technology has been illustrated and described indetail in the drawings and foregoing description, the same is to beconsidered as illustrative and not restrictive in character. It isunderstood that the embodiments have been shown and described in theforegoing specification in satisfaction of the best mode and enablementrequirements. It is understood that one of ordinary skill in the artcould readily make a nigh-infinite number of insubstantial changes andmodifications to the above-described embodiments and that it would beimpractical to attempt to describe all such embodiment variations in thepresent specification. Accordingly, it is understood that all changesand modifications that come within the spirit of the claimed technologyare desired to be protected.

What is claimed is:
 1. A cytometric system for fluorescent radiationconcentration and collection, comprising: a generally hemisphericinternally reflective chamber; a capillary transport system intersectingthe internally reflective chamber; a flow cell positioned in fluidiccommunication with the capillary transport system for storing andreleasing a plurality particles through the capillary transport system;a particle interrogation position located in the chamber; an integrationlaser positioned to shine on the particle interrogation position; atleast one input port formed in the internally reflective chamber; anon-imaging reflector output portion formed operationally connected tothe generally hemispheric internally reflective chamber; and an outputport formed in the non-imaging reflector output portion; wherein theflow cell may be actuated to supply a stream of particles to theparticle interrogation position; and wherein each respective particleoccupying the particle interrogation position is fluoresced by the laserto emit a fluorescent signal to the non-imaging reflector output port.2. The system of claim 1 wherein the surface of the reflective chamberis coated with broadband reflective material.
 3. The system of claim 1and further comprising at least one frequency scattering port formed inthe internally reflective chamber for laser focus, forward scatterdetection, and/or side scatter detection.
 4. The system of claim 1 andfurther comprising a particle transport aid operationally connected tothe capillary transport system, wherein the capillary transport aid isselected from the group including fiber optics, ultrasonic generators,and combinations of thereof.
 5. The system of claim 1 and furthercomprising additional observation ports for imagining of theinterrogation position.
 6. A cytometric system for fluorescent radiationconcentration and collection, comprising: a generally solid, opticallytransparent, at least partially spherical externally reflective chamber;a capillary transport system intersecting the externally reflectivechamber; a flow cell positioned in fluidic communication with thecapillary transport system for storing and releasing a pluralityparticles through the capillary transport system; a particleinterrogation position defined positioned in the chamber; an integrationlaser positioned to shine on the particle interrogation position; anintegration laser positioned to shine on the particle interrogationposition; at least one input port formed in the internally reflectivechamber; a non-imaging reflector output portion formed operationallyconnected to the generally hemispheric internally reflective chamber;and an output port formed in the non-imaging reflector output portion;wherein the flow cell may be actuated to supply a stream of particles tothe particle interrogation position; and wherein each respectiveparticle occupying the particle interrogation position is fluoresced bythe laser to emit a fluorescent signal to the non-imaging reflectoroutput port.
 7. The system of claim 6 wherein the surface of thereflective chamber is coated with broadband anti-reflective material. 8.The system of claim 7 and further comprising at least one frequencyscattering port formed through the broadband anti-reflective materialfor laser focus, forward scatter detection, and/or side scatterdetection.
 9. The system of claim 6 and further comprising a particletransport aid operationally connected to the capillary transport system,wherein the capillary transport aid is selected from the group includingfiber optics, ultrasonic generators, and combinations of thereof. 10.The system of claim 6 and further comprising additional observationports operationally connected to the internally reflective chamber forimagining of the interrogation position.
 11. The system of claim 6wherein the output portion is hyperbolic in shape.
 12. The system ofclaim 6 wherein chamber is spherical in shape.
 13. A cytometric systemfor fluorescent radiation concentration and collection, comprising: agenerally spherical internally reflective chamber; a pixelatedarchitecture substantially enveloping the chamber; a capillary transportsystem intersecting the pixelated architecture and externally into theinternally reflective chamber; a flow cell positioned in fluidiccommunication with the capillary transport system for storing andreleasing a plurality particles through the capillary transport system;a particle interrogation position located in the chamber; a laser inputport operationally connected to the internally reflective chamber; alaser oriented to shine through the laser input port onto the particleinterrogation position; at least one input port formed in the internallyreflective chamber; non-imaging reflector output portion formedoperationally connected to the generally spherical internally reflectivechamber; and an output port formed in the non-imaging reflector outputportion; wherein the flow cell may be actuated to supply a stream ofparticles to the particle interrogation position; wherein eachrespective particle occupying the particle interrogation position may befluoresced to emit a fluorescent signal to the non-imaging reflectoroutput port and captured by the pixelated architecture.
 14. The systemof claim 13 wherein the pixelated architecture further comprises: aplurality of waveguides positioned within the pixelated architecture; atleast one respective input port operationally connected to the pixelatedarchitecture; and at least one respective non-imaging reflector outputport operationally connected to the pixelated architecture; wherein thewavelengths enter the pixelated architecture via the reflector outputport; wherein perspective waveguides are sized to capture wavelengths bysize; and wherein the plurality of uncaptured wavelengths are emittedfrom the non-imagining reflector output port.
 15. The system of claim 14wherein the respective waveguides are composed of consecutive insulator,N, and P layers.
 16. The system of claim 13 and further comprising aparticle transport aid operationally connected to the capillarytransport system, wherein the capillary transport aid is selected fromthe group including fiber optics, ultrasonic generators, andcombinations of thereof.
 17. The system of claim 13 and furthercomprising additional observation ports for imagining of theinterrogation position.